Systems including janus droplets

ABSTRACT

Embodiments described herein may be useful in the detection of analytes. The systems and methods may allow for a relatively simple and rapid way for detecting analytes such as chemical and/or biological analytes and may be useful in numerous applications including sensing, food manufacturing, medical diagnostics, performance materials, dynamic lenses, water monitoring, environmental monitoring, detection of proteins, detection of DNA, among other applications. For example, the systems and methods described herein may be used for determining the presence of a contaminant such as bacteria (e.g., detecting pathogenic bacteria in food and water samples which helps to prevent widespread infection, illness, and even death). Advantageously, the systems and methods described herein may not have the drawbacks in current detection technologies including, for example, relatively high costs, long enrichment steps and analysis times, and/or the need for extensive user training. Another advantageous feature provided by the systems and methods described herein includes fabrication in a relatively large scale. In some embodiments, the systems and methods may be used in conjunction with a detector including handheld detectors incorporated with, for example, smartphones (e.g., for the on-site detection of analytes such as pathogenic bacteria).

CROSS REFERENCE TO RELATED APPLICATIONS

This Application is a Continuation-in-part of U.S. application Ser. No.16/113,520 filed Aug. 27, 2018, entitled “SYSTEMS INCLUDING JANUSDROPLETS”, which is a Continuation of U.S. application Ser. No.15/269,543, filed Sep. 19, 2016, entitled “SYSTEMS INCLUDING JANUSDROPLETS,” each of which is incorporated herein by reference in theirentirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with Government support under Grant No.R01-GM095843 awarded by the National Institutes of Health. TheGovernment has certain rights in the invention.

FIELD OF THE INVENTION

The present invention generally relates to systems and methods includingJanus droplets.

BACKGROUND

Emulsification is a powerful age-old technique for mixing and dispersingimmiscible components within a continuous liquid phase. Consequently,emulsions are central components of medicine, food, and performancematerials. Complex emulsions, including multiple emulsions and Janusdroplets, are of increasing importance in pharmaceuticals and medicaldiagnostics, in the fabrication of microdroplets and capsules for food,in chemical separations, for cosmetics, for dynamic optics, and chemicalseparations. However, quantitative detections of analytes with highsensitivity and selectivity using Janus droplets have yet to berealized. Accordingly, improved systems and methods are needed.

SUMMARY OF THE INVENTION

The present invention provides systems and methods including Janusdroplets.

In one aspect, systems are provided. In some embodiments, the systemcomprises a plurality of Janus droplets associated with binding moietiesto an analyte, the binding moiety and analyte selected such that whenthe analyte binds to the binding moiety at least a portion of theplurality of Janus droplets are changed in orientation sufficient tochange electromagnetic radiation interacting with the plurality of Janusdroplets in a detectable manner.

In some embodiments, the system comprises a plurality of Janus dropletsassociated with a plurality of binding moieties to an analyte and adetector positioned relative to the plurality of Janus droplets suchthat when sufficient numbers of the binding moieties bind to analyte atleast a portion of the plurality of Janus droplets are changed inorientation sufficient to change electromagnetic radiation interactingwith the Janus droplets in a manner determinable by the detector.

In certain embodiments, upon binding to the binding moieties, at least aportion of the plurality of Janus droplets agglutinate.

In certain embodiments, prior to binding to the binding moieties, theplurality of Janus droplets are oriented such that at least a portion ofinterfaces between a first phase and a second phase within each Janusdroplet are aligned parallel with respect to one another.

In certain embodiments, prior to the analyte binding to the bindingmoieties, the plurality of Janus droplets are bound to a surface.

In certain embodiments, upon binding of the analyte to the bindingmoieties, at least a portion of the plurality of Janus droplets unbindfrom the surface.

In certain embodiments, the system comprises a source of external energyapplicable to the composition to generate a determinable signal and adetector positioned to detect the signal.

In certain embodiments, the signal comprises electromagnetic radiation.In certain embodiments, upon exposure of the article to a chemical orbiological analyte, the system generates the determinable signal.

In another aspect, methods are provided. In some embodiments, the methodcomprises allowing an analyte to bind to binding moieties associatedwith a plurality of Janus droplets and determining a change inelectromagnetic radiation interacting with the plurality of Janusdroplets due at least in part to the binding of the analyte to thebinding moieties.

In some embodiments, the method comprises exposing, to an articlecomprising an outer phase and a plurality of Janus droplets dispersedwithin the outer phase, a chemical or biological analyte, wherein thechemical or biological analyte, if present, interacts with at least aportion of the article such that at least a portion of the plurality ofJanus droplets change orientation thereby producing a detectable changein an optical property of the article and determining the detectablechange.

In some embodiments, the method comprises exposing, to an articlecomprising an outer phase and a plurality of Janus droplets dispersedwithin the outer phase, a chemical or biological analyte, wherein thechemical or biological analyte, if present, interacts with at least aportion of the article such that at least a portion of the plurality ofJanus droplets change orientation thereby changing the opticaltransmission of the article.

In certain embodiments, the plurality of Janus droplets comprise one ormore amphiphilic compounds including at least one binding moiety.

In certain embodiments, interacting with at least a portion of thearticle comprises binding of the chemical or biological analyte to theat least one binding moiety.

In certain embodiments, prior to exposing the article to a chemical orbiological analyte, at least a portion of the plurality of Janusdroplets are oriented such that at least a portion of interfaces betweena first phase and a second phase within each Janus droplet are alignedparallel with respect to one another.

In certain embodiments, substantially all of the interfaces between afirst phase and a second phase within each Janus droplet are alignedparallel with respect to one another.

In certain embodiments, upon exposing the article to a chemical orbiological analyte, at least a portion of the plurality of Janusdroplets agglutinate.

In certain embodiments, upon exposing the article to a chemical orbiological analyte, at least a portion of the plurality of Janusdroplets are oriented such that at least a portion of interfaces betweena first phase and a second phase within each Janus droplet are notaligned parallel with respect to one another.

In certain embodiments, at least a portion of the plurality of Janusdroplets are bound to a surface of the article via the binding moiety.

In certain embodiments, upon exposing the article to a chemical orbiological analyte, at least a portion of the plurality of Janusdroplets unbind from the surface.

In yet another aspect, articles are provided. In some embodiments, thearticle comprises an outer phase and a plurality of Janus dropletsdispersed within the outer phase, wherein at least a portion of theplurality of Janus droplets comprise an amphiphilic compound includingat least one binding moiety.

In certain embodiments, the plurality of Janus droplets is oriented suchthat at least a portion of interfaces between a first phase and a secondphase within each Janus droplet are aligned parallel with respect to oneanother.

In certain embodiments, the at least one binding moiety is capable ofbinding with a chemical or biological analyte.

In certain embodiments, upon binding of the at least one binding moietywith a chemical or biological analyte, at least a portion of theplurality of Janus droplets change orientation.

In certain embodiments, the plurality of Janus droplets aresubstantively transmissive to electromagnetic radiation.

In certain embodiments, upon binding of the at least one binding moietywith a chemical or biological analyte, the plurality of Janus dropletsdecrease in optical transmission.

In some embodiments, the article comprises a surface, an outer phasedeposited on at least a portion of the surface, and a plurality of Janusdroplets dispersed within the outer phase, wherein at least a portion ofthe plurality of Janus droplets comprise an amphiphilic compoundincluding at least one binding moiety, and wherein at least a portion ofthe plurality of Janus droplets are bound to the surface via the bindingmoiety.

In certain embodiments, at least a portion of the plurality of Janusdroplets are oriented such that an interface between a first phase and asecond phase within each Janus droplet are not aligned parallel to thesurface.

In certain embodiments, upon exposure of the plurality of Janus dropletsto a biological or chemical analyte, at least a portion of Janusdroplets unbind from the surface.

In certain embodiments, upon exposure of the plurality of Janus dropletsto a biological or chemical analyte, at least a portion of Janusdroplets change orientation.

In certain embodiments, the article is substantively visible-lighttransmissive after exposure to the plurality of Janus droplets to thebiological or chemical analyte.

In certain embodiments, upon exposure of the plurality of Janus dropletsto a chemical or biological analyte, the plurality of Janus dropletsincrease in optical transmission.

In certain embodiments, each Janus droplet comprises a first phase and asecond phase, immiscible with the first phase.

In certain embodiments, the outer phase is an aqueous phase.

In certain embodiments, the first phase comprises a hydrocarbon, afluorocarbon, a silicone, a liquid crystal, an ionic liquid, a polymer,combinations thereof, or derivatives thereof.

In certain embodiments, the second phase comprises a hydrocarbon, afluorocarbon, a silicone, a liquid crystal, an ionic liquid, a polymer,combinations thereof, or derivatives thereof, immiscible with the firstphase.

In certain embodiments, the amphiphilic compound is selected from thegroup consisting of: ionic surfactants, non-ionic surfactants,zwitterionic surfactants, polymers, proteins, DNA, RNA, acids,carbohydrates, saccharides, enzymes, chromophores, lipids, grapheneoxide, combinations thereof, and derivatives thereof.

In an exemplary embodiment, the surface is gallic acid.

In another exemplary embodiment, the surfactant ismaleimide-functionalized polystyrene-b-polyacrylic acid.

In certain embodiments, an interface between the outer phase and theplurality of Janus droplets comprises the amphiphilic compound.

In certain embodiments, the analyte comprises a biological compound, adrug, a macromolecule, a salt, an electrolyte, an enzyme, an acid, anucleic acid, a carbohydrate, a peptide, a protein, a phosphate, asulfonate, a virus, a pathogen, an oxidant, a reductant, a toxin, achemical warfare agent, an explosive, carbon dioxide, or combinationsthereof.

In certain embodiments, the analyte is a single analyte.

In certain embodiments, the analyte is a virus. In some embodiments, thevirus is a zika virus.

In some embodiments, the system comprises a plurality of Janus dropletsassociated with a plurality of binding moieties to a virus and adetector positioned relative to the plurality of Janus droplets suchthat when sufficient numbers of the binding moieties bind to the virusat least a portion of the plurality of Janus droplets are changed inorientation sufficient to change electromagnetic radiation interactingwith the Janus droplets in a manner determinable by the detector.

In some embodiments, the method comprises exposing, to an articlecomprising an outer phase and a plurality of Janus droplets dispersedwithin the outer phase, a sample suspected of containing a virus,wherein the virus, if present, interacts with at least a portion of thearticle such that at least a portion of the plurality of Janus dropletschange orientation thereby producing a detectable change in an opticalproperty of the article and determining the detectable change.

Other advantages and novel features of the present invention will becomeapparent from the following detailed description of various non-limitingembodiments of the invention when considered in conjunction with theaccompanying figures. In cases where the present specification and adocument incorporated by reference include conflicting and/orinconsistent disclosure, the present specification shall control. If twoor more documents incorporated by reference include conflicting and/orinconsistent disclosure with respect to each other, then the documenthaving the later effective date shall control.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A illustrates a system including a Janus droplet, exposed to ananalyte, according to one set of embodiments.

FIG. 1B illustrates a system including a Janus droplet, exposed to ananalyte, according to one set of embodiments.

FIG. 1C illustrates a system including a plurality of Janus droplets,exposed to an analyte, according to one set of embodiments.

FIG. 1D illustrates a system including a plurality of Janus droplets,exposed to an analyte, according to one set of embodiments.

FIG. 2 illustrates a system including a Janus droplet, according to oneset of embodiments.

FIG. 3 illustrates the formation of Janus droplets, according to one setof embodiments.

FIG. 4A shows an exemplary surfactant for use in a system includingJanus droplets, according to one set of embodiments.

FIG. 4B shows the agglutination of a plurality of Janus droplets in thepresence of analyte, according to one set of embodiments.

FIG. 5A shows a monodispersed plurality of Janus droplets, according toone set of embodiments.

FIG. 5B shows a plurality of Janus droplets with altered orientation,according to one set of embodiments.

FIGS. 6A-6B show an exemplary system comprising a plurality of Janusdroplets which, upon exposure to an analyte, changes an optical propertyof the system, according to one set of embodiments.

FIGS. 7A-7F show image processing based of Janus droplets upon exposureto an analyte, according to one set of embodiments.

FIGS. 8A-8F show image processing based of Janus droplets upon exposureto an analyte, according to one set of embodiments.

FIG. 9 shows an illustrative embodiment of interaction with an analyteresulting in the change of orientation of a Janus droplet, according toone set of embodiments.

FIGS. 10A-10B show functionalization of the droplets with the polymersurfactant, according to one set of embodiments. (FIG. 10A) Synthesis ofmaleimide functionalized surfactant P1-MA from apolystyrene-b-polyacrylic acid polymer. (FIG. 10B) Bioconjugation ofrcSso7d to the droplet H/W interface via maleimide-thiol chemistry.

FIG. 11 shows droplets starting at different morphologies, namely H/F/W,Janus and F/H/W functionalized with cysteine engineered rcSso7d,according to one set of embodiments. The continuous phase was exchangedto tune the morphology into the Janus format, followed by the additionof 10 μL of 1 mg mL⁻¹ (0.36 μM) streptavidin. Note that a higher degreeof agglutination is observed with droplets prepared at the higher Tween20 concentrations. The scale bars in the optical micrographs are 100 μm.

FIGS. 12A-12C show optical detection using the backscattering scheme,according to one set of embodiments. (FIG. 12A) Experimental setup withboth excitation and detection source from the top of the emulsion layer.(FIG. 12B) Scheme showing the backscattering of the light with naturallyoriented Janus droplets and agglutinated droplets. (FIG. 12C) Opticalmeasurement with the intensity ratio I_(exc)/I_(H) in correlation to thestreptavidin concentration.

FIGS. 13A-13C show optical scheme using the inner filter effect,according to one set of embodiments. (FIG. 13A) Experimental setup withthe optical fiber at the bottom of the emulsion layer. (FIG. 13B) Schemeshowing the mechanism of the attenuated emission of the perylene dye maydepend, in some cases, on the orientation of the droplets. (FIG. 13C)Experimental data showing the optical detection of the emissionintensity ration I_(H)/I_(F) in correlation of the streptavidinconcentration.

FIG. 14 shows a plot of normalized intensity ratio versus concentrationfor Zika detection using the backscattering and the inner filter effect,according to one set of embodiments.

FIG. 15 is a schematic diagram of backscattering (e.g.,retroreflection), according to one set of embodiments.

Other aspects, embodiments and features of the invention will becomeapparent from the following detailed description when considered inconjunction with the accompanying drawings. The accompanying figures areschematic and are not intended to be drawn to scale. For purposes ofclarity, not every component is labeled in every figure, nor is everycomponent of each embodiment of the invention shown where illustrationis not necessary to allow those of ordinary skill in the art tounderstand the invention. All patent applications and patentsincorporated herein by reference are incorporated by reference in theirentirety. In case of conflict, the present specification, includingdefinitions, will control.

DETAILED DESCRIPTION

Embodiments described herein may be useful in the detection of analytes.The systems and methods may allow for a relatively simple and rapid wayfor detecting analytes such as chemical and/or biological analytes andmay be useful in numerous applications including sensing, foodmanufacturing, medical diagnostics, performance materials, dynamiclenses, water monitoring, environmental monitoring, detection ofproteins, detection of DNA, among other applications. For example, thesystems and methods described herein may be used for determining thepresence of a contaminant such as bacteria (e.g., detecting pathogenicbacteria in food and water samples which helps to prevent widespreadinfection, illness, and even death). Advantageously, the systems andmethods described herein may not have the drawbacks in current detectiontechnologies including, for example, relatively high costs, longenrichment steps and analysis times, and/or the need for extensive usertraining. Another advantageous feature provided by the systems andmethods described herein includes fabrication in a relatively largescale. In some embodiments, the systems and methods may be used inconjunction with a detector including handheld detectors incorporatedwith, for example, smartphones (e.g., for the on-site detection ofanalytes such as pathogenic bacteria). For example, such systems couldbe used by the food industry to prevent extensive foodborne illnesseswhich may result in expensive medical treatment costs, lawsuits,government sanctions, product recalls, and/or tarnished long-termreputations. Articles comprising Janus droplets are also provided.

In some embodiments, the systems and methods comprise a plurality ofJanus droplets. Janus droplets generally include two or more phasesimmiscible with one another and/or having distinct physical and/orchemical properties, within the droplet. In certain embodiments, whenequal amounts of the two immiscible phases are present and theinterfactial tensions are properly balanced, the Janus droplets will bespherical with each hemisphere of the sphere comprising one of theimmiscible phases. In certain embodiments, the plurality of Janusdroplets includes a first phase and a second phase immiscible with thefirst phase. In some embodiments, the plurality of Janus droplets may bedispersed within an outer phase (e.g., an aqueous phase). For example,in some embodiments, the system comprises an aqueous phase and aplurality of Janus droplets comprising a hydrocarbon and a fluorocarbon.In some cases, the plurality of Janus droplets may be associated abinding moiety (e.g., a binding moiety associated with the Janusdroplets and/or a binding moiety present on a surfactant incorporatedwith the plurality of Janus droplets). In some embodiments, the bindingmoiety may bind with an analyte (e.g., a biological and/or chemicalanalyte) such that the orientation of at least a portion of theplurality of Janus droplets is changed. The change in orientation of aJanus droplet may result in a change in the interaction ofelectromagnetic radiation (e.g., visible light) with the Janus dropletin a detectable manner. In some embodiments, exposing a plurality ofJanus droplets to an analyte causes a detectable change in an opticalproperty of the Janus droplets, such that the analyte can be determinedand/or quantified.

In certain embodiments, upon exposure to an analyte, at least a portionof the plurality of Janus droplets may agglutinate. For example, in somecases, the analyte may facilitate the agglutination of at least aportion of the plurality of Janus droplets. The agglutination of someJanus droplets may result in a detectable change in the interaction ofelectromagnetic radiation (e.g., visible light) with the Janus droplets.In some cases, the agglutination of some Janus droplets may result in achange in orientation of each of the Janus droplets (e.g., relative tothe orientation of the Janus droplets prior to exposure to the analyte).In other cases, the Janus droplets may be in a agglutinated state priorto exposure to an analyte and the exposure of the system to the analytewill disrupt agglutination and case a change in the orientation of theJanus droplet.

Advantageously, in some embodiments, the systems described herein mayenable highly sensitive detection of analytes including, for example,detection of single analyte interaction events (e.g., binding events,chemical reactions, biological reactions). In an illustrativeembodiment, a single analyte (e.g., one protein, one strand of DNA, onestrand of RNA) may cause the agglutination of some Janus droplets andchanging the orientation of each of the agglutinated Janus droplets,such that a single analyte (e.g., a single protein, a single strand ofDNA, RNA etc.) is detected. In some such embodiments, the single analytemay bind to some Janus droplets such that the Janus dropletsagglutinate. In another illustrative embodiment, a single analyte maycause the orientation of a single Janus droplet to change (e.g., viaenzymatic degradation of a tether bound to the Janus droplet), such thata single analyte is detected. In some embodiments, a plurality ofanalytes and/or types of analytes may be detected (e.g., via the changein orientation of a plurality of Janus droplets and/or the agglutinationof groups of Janus droplets). In certain embodiments, the concentrationof an analyte exposed to the system may be determined by measuring thenumber of Janus droplets changing orientation upon exposure of thesystem to the analyte.

As illustrated in FIG. 1A, in some embodiments, system 100 comprises aplurality of Janus droplets such as Janus droplet 120. In certainembodiments, Janus droplet 120 comprises first phase 130 (e.g.,comprising a hydrocarbon) and second phase 140 (e.g., comprising afluorocarbon). As depicted illustratively in FIG. 1A, in someembodiments, first phase 130 and second phase 140 may have relativelythe same volume in each Janus droplet. However, those skilled in the artwould understand based upon the teaching of this specification that thevolume of the first phase and the second phase may not be equal.

In some embodiments, as depicted in FIG. 1A, Janus droplet 120 has aparticular orientation, such as orientation 100A. The orientation of aJanus droplet as described herein may be determined by measuring theangle of a planar surface defined by the interface (e.g., interface 125)between the first phase (e.g., first phase 130) and the second phase(e.g., second phase 140). In some embodiments, upon exposure of Janusdroplet 120 to an analyte, the Janus droplet may change orientation(e.g., from orientation 100A to orientation 100B). In some suchembodiments, the analyte may bind with a binding moiety present on theJanus droplet, resulting in the change in orientation of the Janusdroplet. As illustrated in FIG. 1A, the orientation of interface 125 inorientation 100B is different than the orientation of interface 125 inorientation 100A. For example, in some embodiments, the Janus dropletmay rotate upon exposure to the analyte (e.g., upon binding of theanalyte with a binding moiety associated with the Janus droplet). Insome embodiments, the change in orientation of the Janus droplet isdeterminable (e.g., measurable) such that it indicates the presence ofan analyte.

The Janus droplets described herein may be useful in a number ofapplications. In an exemplary embodiment, the Janus droplets describedherein may be used for sensing of an analyte. For example, in some suchembodiments, the Janus droplets may change orientation upon exposure toan analyte such that the change in orientation can be detected (e.g., bya change in optical transmission, polarization, birefringence, etc. ofthe colloid). In another exemplary embodiment, the Janus dropletsdescribed herein may be used as tunable lenses. In certain embodiments,measurements of the optical properties (e.g., transmission, absorption,reflection, focal distance, and scattering) of the Janus droplets can beindicative of specific droplet orientations. For example, when a changein droplet orientation is correlated with an analyte of interest (i.e.,enzyme, pollutant, virus, bacteria, DNA, RNA, etc.), then, the Janusdroplets can be used as sensors in which an optical measurement servesas a readout mechanism of the presence of the analyte. In certainembodiments, for systems in which there is a change in an analyte ofinterest over time (e.g., progress of a chemical reaction, such asdegradation of a chemical by an enzyme over time), tracking of thechanges in optical properties of the Janus droplets over time can beused to, for example, analyze reaction rates or analyte concentrations.In some such embodiments, the orientation of the Janus droplets changesin the presence of an analyte such that the system obtains a transparentstate over a particular range of time, or alternatively, obtains arelatively opaque state over a particular range of time.

Those skilled in the art would understand that changing a property of aJanus droplet refers to a property of the Janus droplet immediatelybefore that differs in a substantially measurable way from the propertyof the Janus droplet at some relatively short time (e.g., seconds,minutes, hours) after exposure to the analyte. Those skilled in the artwould also be capable of selecting methods for determining the change inthe property of the Janus droplets (e.g., measuring the averagebirefringence, measuring the optical transmission at one or morewavelength, measuring the density, etc.) based upon the specificationand examples below.

For example, as illustrated in FIG. 1B, system 102 comprises a pluralityof Janus droplets such as exemplary Janus droplet 120. In someembodiments, electromagnetic radiation 180A interacts with Janus droplet120. In certain embodiments, upon exposure of system 102 to an analyte(e.g., such that the analyte binds to a binding moiety associated withthe Janus droplet), Janus droplet 120 changes orientation (e.g., fromorientation 100A to 100B) sufficiently to change the interaction ofelectromagnetic radiation 180A with the Janus droplets as compared tothe interaction of electromagnetic radiation 180A prior to exposure tothe analyte. For example, prior to exposure to the analyte, Janusdroplet 120 may interact with electromagnetic radiation 180A such thatelectromagnetic radiation 180B is produced. In some embodiments,electromagnetic radiation 180A and electromagnetic radiation 180B may besubstantially the same. For example, Janus droplet 120 may have anorientation 100A such that electromagnetic radiation interacting with(e.g., transmitting perpendicular to interface 125 of Janus droplet 120)is not substantially changed in wavelength and/or amplitude.

For example, in some cases, the plurality of Janus droplets may beorientation such that the system is substantially optically transparentin a direction perpendicular to the surface of the interface between thefirst phase and the second phase (e.g., interface 125). In some cases,however, electromagnetic radiation 180B may be different thanelectromagnetic radiation 180A in wavelength and/or amplitude. In someembodiments, upon exposure of system 102 to an analyte, Janus droplet120 changes orientation from orientation 100A to orientation 100B, suchthat electromagnetic radiation 180A interacts with Janus droplet 120 andproduced electromagnetic radiation 180C, different than electromagneticradiation 180B.

In some embodiments, the plurality of Janus droplets is changed inorientation (e.g., upon exposure to an analyte) sufficient to changeelectromagnetic radiation interacting with the plurality of Janusdroplets in a detectable manner. In certain embodiments, at least aportion of the Janus droplets change orientation thereby changing theoptical transmission of the article and/or thereby producing adetectable change in an optical property of the article. In someembodiments, the detectable change includes a change in color, averageluminescence in one or more directions, and/or average opticaltransmission of the Janus droplet (or system comprising the plurality ofJanus droplets).

In some embodiments the electromagnetic radiation (e.g., theelectromagnetic radiation prior to interacting with the Janus droplet,the electromagnetic radiation after interacting with the Janus droplet)may comprise any suitable wavelength, including but not limited toinfrared light (e.g., a wavelength between about 700 nm and about 1 cm),to visible light (e.g., a wavelength between about 400 nm and about 700nm), and to ultraviolet (UV) light (e.g., a wavelength between about 10nm and about 400 nm).

In certain embodiments, the plurality of Janus droplets (e.g., Janusdroplets 120) is dispersed within an outer phase 110, as illustrated inFIGS. 1A-1C. In some embodiments, the outer phase is an aqueous phase(e.g., comprising water). The aqueous phase may also comprise, in somecases, solutes including organic molecules, proteins, ions, cells, DNA,RNA, cell lysates, or biological organisms. In some embodiments,exposing the system to the analyte comprises introducing the analyteinto the outer phase. In certain embodiments, the analyte may be addedto the outer phase such that the plurality of Janus droplets is exposedto the analyte.

In certain embodiments, the plurality of Janus droplets may be adjacenta surface 150, as illustrated in FIG. 1A. As used herein, when acomponent (e.g., a Janus droplet) is referred to as being “adjacent”another component (e.g., a surface), it can be directly adjacent to thecomponent, or an intervening component (e.g., a fluid) also may bepresent. A component that is “directly adjacent” another component meansthat no intervening component is present (e.g., the component andanother component are in contact with one another). Surface 150 maycomprise a reflective surface such that exposing the system to ananalyte causes a detectable change in an optical property of the Janusdroplets such that the reflected electromagnetic radiation from surface150 is also changed. In an exemplary embodiment, the plurality of Janusdroplets is substantially transparent such that surface 150 is visible(e.g., when viewed perpendicular to surface 150) and, upon exposure toan analyte, the plurality of Janus droplets decrease in opticaltransmission such that at least a portion of surface 150 is obscured.Surface 150 may, in some cases, also be transparent such that light istransmitted through the surface and Janus droplets, such that exposureto an analyte will change the transmission of the light.

In some embodiments, at least a portion of the plurality of Janusdroplets are orientated parallel (e.g., as measuring by the angle of aplanar surface defined by the interface between the first phase and thesecond phase of the Janus droplet) to the surface. For example,referring again to FIG. 1A, in some embodiments, interface 125 of Janusdroplet 120 (prior to exposure to an analyte) is orientatedsubstantially parallel to surface 150 adjacent Janus droplet 120. Incertain embodiments, the plurality of Janus droplets may be orientatedsubstantially parallel to one another (e.g., substantially aligned). Insome embodiments, prior to exposure to an analyte, the plurality ofJanus droplets is aligned/oriented by the force of gravity (e.g., thefirst phase or the second phase having a greater density than the otherphase) such that at least a portion of the plurality of Janus dropletare oriented substantially parallel with one another. In otherembodiments, the forces that cause alignment of Janus droplets mayinclude electrical or magnetic fields. For example, in certainembodiments, the plurality of Janus droplets may include a magneticphase (e.g., including ferromagnetic particles)

In some embodiments, exposure to an analyte results in the agglutinationof a plurality of Janus droplets. For example, as illustrated in FIG.1C, system 104 comprises a plurality of Janus droplets (e.g., exemplaryJanus droplets 120, 122, and 124). In certain embodiments, the pluralityof Janus droplets may be orientated (relative to interfaces 125A, 125B,and 125C) substantially parallel to one another. In some embodiments,the interface between the first phase and the second phase of at least aportion the plurality of Janus droplet is aligned normal to the primarydirection of the force of gravity such that the plurality of Janusdroplets are oriented substantially parallel to one another. In someembodiments, upon exposure to an analyte, at least a portion of theJanus droplets agglutinate. In certain embodiments, agglutination of theJanus droplets results in a change of orientation of at least a portionof the Janus droplets (e.g., as measured by the change in angle ofinterfaces 125A, 125B, and 125C).

In certain embodiments, a binding moiety associated with the Janusdroplet may bind with the analyte such that the Janus dropletsagglutinate. For example, referring again to FIG. 1C, upon exposure toan analyte, the analyte may bind to a binding moiety on two or moreJanus droplets (e.g., forming a bound complex 150 between two or moreJanus droplets such as between Janus droplet 120 and Janus droplet 122).One of ordinary skill in the art would understand, based upon theteachings of this specification, that while bound complex 150 isillustrated as binding between first phase 130 and second phase 140,that formation of a bound complex between first phase 130 and firstphase 130 of two droplets, is also possible. For example, as shownillustratively in FIG. 15, droplet 120 and droplet 122 are agglutinatedvia bound complex 152 between first phase 130 of droplet 120 and firstphase 130 of droplet 122. Other configurations are also possible.

In some embodiments, a plurality of binding moieties (e.g., bindingmoieties associated with one or more Janus droplets) may bind with oneor more analytes mutlivalently. For example, as illustrated in FIG. 1D,analyte 155 binds multivalently with Janus droplet 120, Janus droplet122, and Janus droplet 124 such that the Janus droplets agglutinate. Insome such embodiments, upon exposure and binding to the analyte, theJanus droplets change orientation sufficient to change electromagneticradiation interacting with the plurality of Janus droplets in adetectable manner.

In some embodiments, upon agglutination of two or more Janus droplets,at least a portion of incident electromagnetic radiation mayretroreflect amongst the droplets such that at least a portion of theelectromagnetic radiation is reflected. For example, as shownillustratively in FIG. 15, system 106 comprises a plurality of Janusdroplets (e.g., exemplary Janus droplets 120 and 122). In certainembodiments, the plurality of Janus droplets may be orientated (relativeto interfaces 125A, and 125B) substantially parallel to one another(100C) and such that electromagnetic radiation 160 is transmittedthrough the interfaces. In some embodiments, upon exposure to ananalyte, at least a portion of the Janus droplets agglutinate (100D). Incertain embodiments, agglutination of the Janus droplets results in achange of orientation (100D) of at least a portion of the Janus droplets(e.g., as measured by the change in angle of interfaces 125A and 125B).In some embodiments, the Janus droplets change angle such that at leasta portion of electromagnetic radiation 160 is reflected off ofinterfaces 125A and 125B. In some embodiments, at least a portion ofelectromagnetic radiation may still transmit through system 106. In someembodiments, the portion of electromagnetic radiation 160 that isreflected may be detected (e.g., by an optical detector, by a user)indicating the presence of the analyte (e.g., the analyte that resultsin agglutination of the Janus droplets) in the system.

In certain embodiments, the system may comprise a plurality of Janusdroplets tethered (e.g., bound) to a surface. In some embodiments,exposure of the system to an analyte results in the breaking (e.g.,cleavage) of the tether such that at least a portion of the Janusdroplets change orientation (e.g., sufficient to change electromagneticradiation interacting with the plurality of Janus droplets in adetectable manner). For example, as illustrated in FIG. 2, system 200comprises Janus droplet 220 comprising first phase 230 and second phase240, tethered to surface 260 adjacent Janus droplet 220 via tether 270.In some embodiments, exposure to an analyte results in the breaking oftether 270 such that Janus droplet 220 changes orientation (fromorientation 200A prior to exposure to the analyte to orientation 200Bupon exposure to the analyte). Those skilled in the art would understandbased upon the teachings of this specification that surface 260 need notbe planar and could be, for example, curved (e.g., the surface comprisesa polymeric and/or inorganic particle). In some cases the surface mayinclude an assembly of molecules such as proteins, DNA or RNA. Incertain embodiments, the surface may comprise biological tissue (e.g.,comprising skin (e.g., human skin), organ tissues, cells, or the like).In some cases, the surface may be a liquid immiscible with the outerphase and/or one or more phases present within the Janus droplets. Insome embodiments, the surface comprises a polymeric material.

In some embodiments, the Janus droplet is tethered to the surface suchthat the interface between the first phase and the second phase is notparallel to the adjacent substrate and/or is not parallel with at leasta portion of the plurality of Janus droplets. In some such embodiments,upon breaking of the tether by the analyte, at least a portion of theJanus droplets change orientation (e.g., such that at least a portion ofthe Janus droplets are parallel with one another and/or are parallelwith an adjacent substrate). In some cases, breaking of the tether bythe presence of an analyte resulting in an increase in the opticaltransmission of the system (e.g., such that a feature on the substrateis visible when viewed perpendicular to the surface). The tether mayinclude, for example, one or more proteins, a polymer, one or morestrands of DNA, one or more strands of RNA, or combinations thereof.Other tethers are also possible.

The analyte may break the tether in any suitable manner. For example, insome embodiments, the analyte may cleave the tether (e.g., via enzymaticdegradation). In certain embodiments, the analyte may cleave the tetherby changing the pH of the outer phase such that the tether breaks. Insome embodiments, the analyte may cause the cleavage of the tether suchthat one or more binding moieties associated with (e.g., integratedwithin) the plurality of Janus droplets bind to the analyte. In somesuch embodiments, one or more binding moieties may be bound to thetether such that the Janus droplet is bound to the surface and, uponexposure to the analyte, the binding moiety unbinds from the tether andbinds to the analyte.

In some cases, the binding moiety may comprise a biological or achemical group capable of binding another biological or chemicalmolecule in a medium (e.g., aqueous phase). For example, the bindingmoiety may include a functional group, such as a thiol, aldehyde, ester,carboxylic acid, hydroxyl, and the like, wherein the functional groupforms a bond with the analyte. In some cases, the binding moiety may bean electron-rich or electron-poor moiety wherein interaction between theanalyte and the binding moiety comprises an electrostatic interaction.In some cases, the interaction between the analyte and the bindingmoiety includes binding to a metal or metal-containing moiety.

In some embodiment, the binding moiety and analyte interact via abinding event between pairs of biological molecules including proteins,nucleic acids, glycoproteins, carbohydrates, hormones, drugs, and thelike. Specific examples include an antibody/peptide pair, anantibody/antigen pair, an antibody fragment/antigen pair, anantibody/antigen fragment pair, an antibody fragment/antigen fragmentpair, an antibody/hapten pair, an enzyme/substrate pair, anenzyme/inhibitor pair, an enzyme/cofactor pair, a protein/substratepair, a nucleic acid/nucleic acid pair, a protein/nucleic acid pair, apeptide/peptide pair, a protein/protein pair, a small molecule/proteinpair, a glutathione/GST pair, an anti-GFP/GFP fusion protein pair, aMyc/Max pair, a maltose/maltose binding protein pair, acarbohydrate/protein pair, a carbohydrate derivative/protein pair, ametal binding tag/metal/chelate, a peptide tag/metal ion-metal chelatepair, a peptide/NTA pair, a lectin/carbohydrate pair, a receptor/hormonepair, a receptor/effector pair, a complementary nucleic acid/nucleicacid pair, a ligand/cell surface receptor pair, a virus/ligand pair, aProtein A/antibody pair, a Protein G/antibody pair, a Protein L/antibodypair, an Fc receptor/antibody pair, a biotin/avidin pair, abiotin/streptavidin pair, a drug/target pair, a zinc finger/nucleic acidpair, a small molecule/peptide pair, a small molecule/protein pair, asmall molecule/target pair, a carbohydrate/protein pair such asmaltose/MBP (maltose binding protein), a small molecule/target pair, ora metal ion/chelating agent pair. Specific non-limiting examples ofbinding moieties include peptides, proteins, DNA, RNA, PNA. Otherbinding moieties and binding pairs are also possible. Binding moietiescan also be attached to polymers, organic nanoparticles, inorganicnanoparticles, or metal nanoparticles.

In some embodiments, the binding moiety and the tether interact via abinding event between pairs of biological molecules including proteins,nucleic acids, glycoproteins, carbohydrates, hormones, and the like. Inother embodiments the binding moieties can be also bound to ananoparticle.

In an exemplary embodiment, the binding moiety comprises a protein. Insome embodiments, the protein is a hyperthermophilic protein.

The analyte may comprise any suitable material (e.g., a vapor analyte, aliquid analyte, a solid analyte) such that the incorporation of theanalyte into the system causes at least a portion of the plurality ofJanus droplets to change orientation (e.g., via breaking of a tetherand/or agglutination of the Janus droplets). Those skilled in the artwould be capable of selecting analytes and components suitable for Janusdroplets based upon the teaching of the specification and the examplesbelow. Non-limiting examples of analytes include a biological compound,a drug, a macromolecule, a salt, an electrolyte, an enzyme, a nucleicacid, a carbohydrate, a peptide, a protein, a lipid, a phosphate, asulfonate, a virus, a pathogen (e.g., bacteria, virus), an oxidant, areductant, a toxin, a chemical warfare agent, an explosive, carbondioxide, a surfactant, or combinations thereof. In some embodiments, thetether is a biological compound, a drug, a macromolecule, a salt, anelectrolyte, an enzyme, a nucleic acid, a carbohydrate, a peptide, aprotein, a lipid, a phosphate, a sulfonate, a virus, a pathogen, anoxidant, a reductant, a toxin, a chemical warfare agent, an explosive,carbon dioxide, a surfactant, or combinations thereof. In an exemplaryembodiment, the analyte is a bacterium.

In another exemplary embodiments, the analyte is a virus. In someembodiments, the virus is a zika virus.

In an exemplary embodiment, an enzyme may be added to the systemcomprising a plurality of Janus droplets such that the enzyme interactswith one or more of the components, binding moieties, tethers, and/oramphiphilic compounds present in the plurality of Janus droplets. Insome such embodiments, the enzyme may interact with the component,binding moiety, tether, and/or amphiphilic compound (e.g., such as asurfactant which is cleaved in the presence of the enzyme) such that atleast a portion of the plurality of Janus droplets change orientation asdescribed herein. In certain embodiments, the Janus droplets changeorientation at a particular critical concentration of the analyte.

In another exemplary embodiment, one or more Janus droplets may comprisean amphiphilic compound such as a surfactant that is capable ofinteracting with a biological analyte. In some such embodiments, theJanus droplet may change orientation in the presence of a biologicalanalyte such that the change in orientation can be detected (e.g., byoptical transmission).

In some embodiments, the interaction between a binding moiety and theanalyte includes a chemical transformation between the binding moietyand the analyte and/or the binding moiety and a tether. Non-limitingexamples of chemical transformations include enzymatic degradation,enzymatic synthesis, ionization, cleavage, coupling, hybridization,aggregation, hydrolysis, isomerization, reduction, oxidation, andhost-guest interactions of one or more components (or componentmaterials such as a surfactant). Other chemical transformations are alsopossible.

As described herein, in some embodiments, the methods and systemscomprise an outer phase and a plurality of Janus droplets dispersedwithin the outer phase. In certain embodiments, the plurality of Janusdroplets comprises two or more phases. The two or more phases (e.g., afirst phase and a second phase) may be substantially miscible over arange of temperatures (e.g., below a critical temperature, above acritical temperature). The two or more phases may also be substantiallyimmiscible over a different range of temperatures (e.g., above thecritical temperature, below the critical temperature) than the range oftemperatures over which they are miscible. The use of two or more phaseswith differing miscibility at different temperatures may allow for theone-step formation (e.g., bulk) of such Janus droplets, unconstrained bythe limits of previous methods (e.g., low yield of microfluidic devices,multi-step processes, the need for solvent addition and/or extraction,etc.).

Janus droplets described herein may be formed using any suitable method.For example, in some embodiments, an outer phase material, a firstphase, and a second phase are mixed and emulsified, forming an outerphase and a plurality of Janus droplets dispersed within the outerphase. Suitable methods for emulsifying the fluid are known in the artand may comprise sonication, high shear mixing, shaking, passing thefluid through a membrane, or injecting the two or more components intothe outer phase through a small diameter channel.

Non-limiting examples of methods for forming Janus droplets aredescribed in more detail in commonly-owned U.S. Patent PublicationNumber 2016/0151753, entitled “Compositions and Methods for FormingEmulsions”, filed Oct. 30, 2015 and in U.S. Patent Publication Number2016/0151756, entitled “Compositions and Methods for Arranging ColloidPhases”, filed Oct. 30, 2016, each of which is incorporated herein byreference in its entirety.

Immiscible, as used herein, refers to two phases having an interfacialtension of greater than or equal to 0.01 mN/m as determined by aninverted pendant drop goniometer. Conversely, miscible, as used herein,refers to two phases having an interfacial tension of less than 0.01mN/m as determined by an inverted pendant drop goniometer.

The term phase, as used herein, generally refers to a portion of adroplet or fluid comprising a group of substantially similar molecules,and/or a group of substantially similar compounds. Those skilled in theart would understand that is not intended to refer to single moleculesor atoms. In some embodiments, the phase is a liquid phase (e.g., anaqueous phase, a non-aqueous phase) comprising a group of substantiallysimilar compounds and/or molecules and/or polymers. For example, in somecases, each phase may occupy at least about 1 vol %, at least about 2vol %, at least about 5 vol %, at least about 10 vol %, at least about20 vol %, at least about 50 vol %, at least about 70 vol %, at leastabout 90 vol %, at least about 95 vol %, or at least about 99 vol % ofthe total volume of the two or more phases.

In some embodiments, at least one of the two or more phases (e.g., thefirst phase) comprises a hydrocarbon. Non-limiting examples of suitablehydrocarbons include alkanes (e.g., hexane, heptane, decane, dodecane,hexadecane), alkenes, alkynes, aromatics (e.g., benzene, toluene,xylene, benzyl benzoate, diethyl phalate), oils (e.g., natural oils andoil mixtures including vegetable oil, mineral oil, and olive oil),liquid monomers and/or polymers (e.g., hexanediol diacrylate, butanedioldiacrylate, polyethylene glycols, trimethylolpropane ethoxylatetriacrylate), alcohols (e.g., butanol, octanol, pentanol), ethers (e.g.,diethyl ether, diethylene glycol, dimethyl ether), nitromethane,halogenated liquids (e.g., chloroform, dichlorobenzene, methylenechloride, carbon tetrachloride), brominated liquids, iodinated liquids,lactates (e.g., ethyl lactate), acids (e.g., citric acid, acetic acid),liquid crystals (4-cyano-4′-pentylbiphenyl), trimethylamine, liquidcrystal hydrocarbons (e.g., 5-cyanobiphenyl), combinations thereof, andderivatives thereof, optionally substituted. In some embodiments, thehydrocarbon comprises a halogen group, sulfur, nitrogen, phosphorous,oxygen, or the like. Other hydrocarbons and solutes are also possible.

In some embodiments, at least one of the two or more phases (e.g., thesecond phase) comprises a fluorocarbon. Non-limiting examples ofsuitable fluorocarbons include fluorinated compounds such asperfluoroalkanes (e.g., perfluorohexanes, perfluorooctane,perfluorodecalin, perfluoromethylcyclohexane), perfluoroalkenes (e.g.,perfluorobenzene), perfluoroalkynes, and branched fluorocarbons (e.g.,perfluorotributylamine). Additional non-limiting examples of suitablefluorocarbons include partially fluorinated compounds such asmethoxyperfluorobutane, ethyl nonafluorobutyl ether,2H,3H-perfluoropentane, trifluorotoluene, perfluoroidodide, fluorinatedor partially fluorinated oligomers,2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9-hexadecafluorodecane-1,10-diylbis(2-methylacrylate), perfluoroiodide, and2-(trifluoromethyl)-3-ethoxydodecafluorohexane. Other fluorocarbons arealso possible.

In some embodiments, at least one of the two or more components phases asilicone such as silicone oil. Non-limiting examples of suitablesilicone oils include polydimethylsiloxane and cyclosiloxane fluids.

In some embodiments, at least one of the two or more phases compriseswater.

In some embodiments, at least one of the two or more phases comprises anionic liquid (e.g., an electrolyte, a liquid salt). In some embodiments,at least one of the two or more inner phases comprises an ionic liquid(e.g., an electrolyte, a liquid salt, 1-allyl-3-methylimidazoliumbromide, 1-allyl-3-methylimidazolium chloride,1-benzyl-3-methylimidazolium hexafluorophosphate,1-butyl-1-methylpyrrolidinium hexafluorophosphate). In some embodiments,the outer phase comprises water. In certain embodiments, at least one ofthe two or more phases comprises a deuterated compound (e.g., adeuterated hydrocarbon).

In some embodiments, at least one of the two or more phases comprises achlorinated solvent (e.g. chloroform, carbon tetrachloride).

In some embodiments, at least one of the two or more phases comprises acombination of the materials described above (e.g., comprising ahydrocarbon, a fluorocarbon, a silicone, or combinations thereof).Non-limiting examples of combinations of phases present in the Janusdroplets described herein include hexane and perfluorohexane, carbontetrachloride and perfluorohexane, chloroform and perfluorohexane,hexane and perfluorodecalin, hexane and perfluoromethylcyclohexane,hexane and perfluorotributylamine, isopropanol and hexadecane, ethyllactate and heptane, acetic acid and decane, and triethylamine andwater. Other combinations and materials are also possible.

Those skilled in the art would be capable of selecting suitable phasesbased upon the teachings of the specification and the examples belowsuch that the two or more phases are immiscible under a particular rangeof temperatures and/or conditions, as described above.

The outer phase may comprise any suitable material. Generally, the twoor more phases comprising the plurality of Janus droplets may besubstantially immiscible with the outer phase. In some embodiments, theouter phase is an aqueous phase (e.g., comprising water). The aqueousphase may, in some cases, have ions and/or be mixed with a biologicalfluid (e.g., sputum, blood, plasma, urine). In certain embodiments, theouter phase is a non-aqueous phase. In some embodiments, the non-aqueousphase comprises a hydrocarbon, a fluorocarbon, a silicone, or the like,as described above in the context of the two or more phases,substantially immiscible with the two or more phases. Those skilled inthe art would be capable, based upon the teachings of the specificationand the examples below, of selecting suitable materials for use as anouter phase based upon the miscibility of those materials (e.g., suchthat the two or more phases are substantially immiscible with the outerphase). The use of a non-aqueous outer phase may be advantageous incertain applications where the emulsion is used in low humidityenvironments. For example, a plurality of Janus droplets comprisingfluorocarbon/hydrocarbon phases can be created in a liquid siliconematrix.

In some embodiments, the Janus droplet comprises an amphiphiliccompound. In certain embodiments, the binding moiety is associated withthe amphiphilic compound. For example, the binding moiety may be boundto at least a portion of the amphiphilic compound.

In certain embodiments, the amphiphilic compound is miscible in theouter phase. In some embodiments, the amphiphilic compound is misciblein at least one of the two or more phases (e.g., the first phase, thesecond phase). In certain embodiments, the amphiphilic compound has agreater miscibility in at least one of the two or more phases than amiscibility in the outer phase. In other embodiments the amphiphiliccompound is added to the Janus droplet though a dispersion, such as anaqueous micelle structure or dissolution method (e.g., comprisinginjecting a dispersion of the amphiphilic compound into the solutioncontaining the Janus droplets). In some embodiments, the amphiphiliccompound is disposed at the interface between the outer phase and theplurality of Janus droplets. Amphiphilic compounds may also begenerated, in some embodiments, by reaction of a solute in one phasewith solute in another phase. For example, without wishing to be boundby theory, a reactive group in an organic phase may, in some cases,react with a solute from an aqueous phase to create a amphiphilicmolecule at the surface of a droplet. In certain embodiments, theamphiphilic compound is disposed at the interface between at least twoof the two or more phases (e.g., the interface between the first phaseand the second phase). The amphiphilic compound may preferentiallyinteract with one or more phases or the outer phase. Those skilled inthe art would be capable of selecting a suitable amphiphilic compoundbased upon the teachings of the specification and examples below.

In some embodiments, the amphiphilic compound is a surfactant.Non-limiting examples of suitable surfactants include ionic surfactants,non-ionic surfactants, and zwitterionic surfactants. In someembodiments, the surfactant is a fluorosurfactants (e.g., commerciallyavailable fluorosurfactants such as Zonyl® or Capstone®). In certainembodiments, the surfactant is anionic surfactants (e.g., sodium dodecylsulfate (SDS)), cationic surfactants (e.g., alkyltrimethyl ammoniumchloride, alkylmethyl ammonium bromide), non-ionic surfactants (e.g.,alkyl poly(ethylene oxide)), zwitterionic surfactants (e.g., alkylbetain, C8-lecitin), polymeric surfactants, gemini surfactants,particulate surfactants (e.g., graphene oxide, silica particles, goldnanoparticles, polymer nanoparticles), and combinations thereof. Othersurfactants are also possible. In some embodiments, the amphiphiliccompound is a nucleic acid (e.g., DNA, RNA). In certain embodiments theamphiphilic compound comprises an amino acid (e.g., a peptide, aprotein). In some embodiments, the amphiphilic compound comprises abiomaterial. Non-limiting examples of suitable biomaterials includecarbohydrates or derivatives thereof, saccharides or derivatives thereof(e.g., sialic acid), lipids or derivatives thereof, enzymes,chromophores or the like. Those skilled in the art would be capable ofselecting suitable biomaterials based upon the teachings of thespecification and the examples below.

In some embodiments, the amphiphilic compound comprises a perfluorinatedsegment. In some embodiments, the amphiphilic compound comprisesethylene glycol.

In some embodiments, the amphiphilic compound is capable of formingmetal complexes.

In some embodiments, the amphiphilic compound is gallic acid. In someembodiments, the amphiphilic compound comprisespolystyrene-b-polyacrylic acid or a derivative thereof.

In some embodiments, the one or more phases (e.g., the first phase, thesecond phase) and/or the outer phase comprises an additional compounddispersed in the one or more phases and/or the outer phase. In certainembodiments, the additional compound is miscible/dispersible in thefirst phase and immiscible/not dispersible in the second phases. In somecases, at least a portion of the additional compound is dispersible inthe first phases and not dispersible in the second phases (e.g., asurfactant). In some embodiments, the additional compound may bedispersible or not dispersible in the outer phase. Non-limiting examplesof suitable additional compounds include particles (e.g., magneticparticles/nanoparticles, silica particles), biological molecules (e.g.,insulin), pharmaceutical compounds, polymers, surfactants, cells,bacteria, viruses, active pharmaceutical ingredients, and metals ormetal particles. Other additional compounds are also possible and thoseskilled in the art would be capable of selecting such compounds basedupon the teachings of this specification.

In some embodiments, the plurality of Janus droplets can be formed byadjusting the temperature of a fluid comprising the outer phase and thetwo or more immiscible phases such that the two or more phases becomesubstantially miscible with each other, and emulsifying the fluid (e.g.,thus forming the plurality of Janus droplets). In certain embodiments,the method comprises adjusting the temperature of the fluid comprisingthe two phases such that the two or more phases become substantiallyimmiscible. In other embodiments, the method comprises the addition of asolvent that creates a stable uniform composition prior toemulsification, and the solvent is removed by evaporation or extractionto give phase separation and produce a Janus droplet.

For example, as illustrated in FIG. 3, a fluid 300A comprises firstphase 310 (e.g., a hydrocarbon) and second phase 320 (e.g., afluorocarbon) which are immiscible at a first temperature T₀. In someembodiments, T₀ is adjusted to a second temperature T₁ (e.g., where T₁is greater than T₀, or where T₁ is less than T₀) such that the firstcomponent and second component form a miscible mixture 330 in fluid300B. For example, in some embodiments, the first phase and the secondphase, which are initially substantially immiscible, may be heated suchthat they are miscible. In certain embodiments, the first phase and thesecond phase, which are initially substantially immiscible, may becooled such that they are miscible. Miscible mixture 330 can, in certainembodiments, be emulsified to form emulsion 300C comprising plurality ofdroplets 332. Plurality of droplets 332 may comprise miscible mixture330 and be present in an outer phase 340. In some cases, outer phase 340may be added prior to changing the temperature from T₀ to T₁. In certainembodiments, outer phase 340 may be added after changing the temperaturebut prior to emulsification.

In some embodiments, T₁ is adjusted to a temperature T₂ (e.g., where T₂is greater than T₁ or where T₂ is less than T₁) such that droplet 332comprises first phase 310, and second phase 320 substantially immisciblewith first component 310, forming a Janus droplet.

In some embodiments, T₁ is greater than a critical temperature of thetwo or more phases (e.g., an upper consolute temperature of the two ormore phases). In certain embodiments, T₁ is less than a criticaltemperature of the two or more phases (e.g., a lower consolutetemperature). Those skilled in the art will be capable of selectingsuitable methods for determining the critical temperature (e.g., theupper consolute temperature, the lower consolute temperature) of two ormore phases.

Suitable methods for emulsifying the fluid are known in the art and maycomprise sonication, high shear mixing, shaking, passing the fluidthrough a membrane, or injecting the two or more components into theouter phase through a small diameter channel.

The plurality of Janus particles may have any suitable averagecross-sectional dimension. In some embodiments, the averagecross-sectional dimension of the plurality of Janus particles is greaterthan or equal to 400 nanometers, greater than or equal to 500nanometers, greater than or equal to 600 nanometers, greater than orequal to 800 nanometers, greater than or equal to 1 micron, greater thanor equal to 2 microns, greater than or equal to 5 microns, greater thanor equal to 10 microns, greater than or equal to 20 microns, greaterthan or equal to 30 microns, greater than or equal to 50 microns,greater than or equal to 60 microns, greater than or equal to 75microns, greater than or equal to 100 microns, greater than or equal to150 microns, greater than or equal to 200 microns, greater than or equalto 300 microns, or greater than or equal to 400 microns. In certainembodiments, the average cross-sectional dimension of the plurality ofJanus particles may be less than or equal to 500 microns, less than orequal to 400 microns, less than or equal to 300 microns, less than orequal to 200 microns, less than or equal to 150 microns, less than orequal to 100 microns, less than or equal to 75 microns, less than orequal to 60 microns, less than or equal to 50 microns, less than orequal to 20 microns, less than or equal to 10 microns, less than orequal to 5 microns, less than or equal to 2 microns, less than or equalto 1 micron, less than or equal to 800 nanometers, less than or equal to600 nanometers, or less than or equal to 500 nanometers. Combinations ofthe above-referenced ranges are possible (e.g., greater than or equal to400 nanometers and less than or equal to 500 microns, greater than orequal to 400 nanometers and less than or equal to 100 microns, greaterthan or equal to 30 microns and less than or equal to 200 microns).Other ranges are also possible.

EXAMPLES

The following examples illustrate embodiments of certain aspects of theinvention. It should be understood that the methods and/or materialsdescribed herein may be modified and/or scaled, as known to those ofordinary skill in the art.

The following examples demonstrate the use of systems for the detectionof analytes.

Surfactants specially designed with recognition elements to bindtargeting analytes (species/molecules of interest) multivalently weresynthesized. The binding interaction was able to transform a pluralityof Janus droplets from an upright position to a horizontally tiltedposition against gravity. This transformation generated a distinctoptical signal (scattering of a light beam) in the presence of analytes.The opposite response was also possible wherein a plurality of Janusdroplets were pre-titled by binding to a surface or particle and isinitially in a scattering position. In this case, the action of ananalyte was to disrupt the linkage between the surface or particle andallow a relaxation to an upright position that allowed for reducedscattering. The optical signal could be recorded via a smartphone by forexample using a QR code for binary on/off detection, using lowmagnification images that are processed computationally to quantify theamount of analytes in the emulsion mixture, and/or the monitoring thetransmission of focused light beams through the samples. Such systemscould be used in biosensor applications including aqueous liquid phasedetection. The emulsions (comprising Janus droplets) with only lowmolecular weight surfactant molecules were relatively inexpensive tofabricate and stable over multiple weeks with no further precautions. Incases where greater emulsion stability may be required, polymericsurfactant molecules and structures could be employed. Additionally, theJanus droplets were highly selective and sensitive for detection ofpathogens as, in some cases, small changes in the concentration and/orthe identity of the surfactants lead to significant changes in theorienation of the Janus droplets. Janus droplets were fabricated usingeither bulk emulsification, which generated polydisperse droplets, or ina microfluidic device, which generated monodisperse droplets. Forsurfactants soluble in water, a solution containing the functionalizedsurfactants was used as the continuous phase. Hydrocarbon phase (such ashexane, ortho-dichlorobenzene, phthalate, etc.) and fluorocarbon phase(such as perfluorohexane, ethyl nonafluorobutyl ether, methoxyperfluorobutane) were mixed and heated over the upper criticaltemperature to generate the single droplet phase. When the droplet phasewas dispersed into the continuous (outer) phase containing surfactants,single emulsions were generated; and upon cooling, the hydrocarbon andfluorocarbon phases separated to generate Janus droplets. Thecomposition of each droplet was substantially similar because they weregenerated from the same single droplet phase. In addition, surfactantswere able to be incorporated into the droplet phase. Surfactants thatwere not soluble in water were dissolved in the hydrocarbon phase or thefluorocarbon phase before mixing. The droplet phase containingsurfactants could then be dispersed into the continuous water phase,which may contain additional surfactants and surfactant assemblies togenerate the droplets. In both cases, Janus droplets were used assensing particles with surfaces covered by with functionalizedsurfactants. The surfactants or surfactant assemblies could containpolymer surfactant/stabilizers or macromolecules of biologicalsignificance, including proteins, enzymes, nucleic acids, DNA, RNA.

Sensing of Pathogenic Bacteria

Our approach to detect pathogenic bacteria took advantage of the generalaffinity that different bacteria exhibit for specific patterns ofcarbohydrate. One of the targeting analytes, Escherichia coli (E. coli),is a bacterium that can be easily spread in contaminated food and water.While most strains of E. coli are harmless, certain strains that producetoxins could cause serious and fetal illness. To detect the E. Colibacteria, surfactants were carefully designed that interact with thesurfaces of the cell via the carbohydrate-lectin interaction. This weakinteraction between lectin on the surfaces of E. coli and D-mannosetypically creates a challenge to detect bacteria with high sensitivitywhen relying on a single interaction. Thus, a surfactant thatfunctionalizes one phase of the Janus droplets to increase theconcentration of the mannose moiety on the surface was designed. Theincrease in the concentration of the mannose moiety significantlyenhanced binding affinity between the bacteria and the droplets,transforming a droplet into a selective sensing particle. The bindingbetween Concanavalin A (ConA), a lectin known to bind D-mannose, wasinitially investigated using the Janus droplets as a model system. Thistechnology could be relatively easily adapted for other analytes by, forexample, changing the active surfactants. A novel surfactant bearing aD-mannose head group (ManC14) was synthesized (FIG. 4A). FIG. 4A showsthe scheme for Mannose surfactant (ManC14) synthesis. FIG. 4B shows aschematic illustration of Janus droplets aligning with Concanavalin A(ConA). The denser perfluorohexane phase aligned at the bottom and thehexane on the top of the Janus droplets.

For this particular sensing platform, the Janus droplets were fabricatedusing the following method. The surfactants ManC14 and Zonyl® FS 300 (acommercially available fluorocarbon surfactant) were dissolved in aHEPES buffer solution (pH=7.5) as the continuous phase. A mixture ofhexane and perfluorohexane (single droplet phase) was dispersed into thesurfactant solution and cooled down to generate Janus droplets. Thehexane phase on the Janus droplets was functionalized with mannosegroups where the surfactant ManC14 aligned preferentially at thehexane/water interface. Without wishing to be bound by theory, due togravity and the higher density of perfluorohexane in relative to that ofhexane, Janus droplets aligned with perfluorohexane phase in the bottom(FIG. 4B). ConA was dissolved in HEPES buffer solution with finalconcentration of 0.5 mg mL⁻¹. An increasing amount of this solution (10μL to 40 μL) was added to the Janus droplets; and after swirling thesolution, the two-faced Janus droplets started aligning in a uniquetilted configuration. The surfaces that were stabilized by ManC14surfactant agglutinated together to form droplet complexes (FIG. 4B).

Without wishing to be bound by theory, the agglutination phenomenonoccured because ConA has four subunits, each with a binding site formannose. This four-site binder acted similarly to an antibody that bindsmultiple particles and joins them together to make agglutinated dropletcomplexes. When Janus droplets agglutinate, the solution changes fromtransparent to opaque. This large and easily observable change isparticularly powerful because detection events will not generallyrequire, for example, any external power input. The Janus dropletagglutination level could be characterized both qualitatively andquantitatively as described herein.

Tuning the Surface Chemistry

Surface recognition is a general phenomenon that can be applied to manydifferent types of methods. The use of a ligand surfactant binding witha multivalent receptor, which can be a protein, cell, or pathogen,nanoparticle was described above. This scheme can be reversed where areceptor is immobilized at the surface of a droplet and then use amultivalent ligand scaffold (natural or synthetic) to bind the Janusdroplets and hold them in a tilted (scattering) state relative to thealigned non-scattering state favored by gravity. The ligands can bedesigned to have a lower affinity than a target analyte and henceexposure to the analyte can result in a displacement that breaks thelinkage (e.g., tether) between the polyvalent ligand and the droplet.Similarly, the tether between the droplet and the ligand can be cleaved.This could be affected by an enzyme that cleaves a peptide, such as anester or a degraded RNA. It could also be affected by catalytic or heavymetal ions or select nucleophiles (sulfides). In some cases, the ligandscould be bound to a surface. It is also possible that the ligands resideon another droplet.

Individual droplets that are tilted or alternatively not tilted (alignedby gravity) can be relatively easily quantified. This gives rise to theability to, in some cases, detect single analytes. For example, it ispossible that a single molecule of DNA can be detected if the droplet isanchored to a surface by a DNA duplex. Disruption of this duplex by acomplementary target DNA analyte can be observed. One aligned droplet ina sea of other tilted droplets would be readily detected. This schemehas an advantage that, for example, there would be many potentialbinding sites for the DNA molecule and hence thus the target DNA wouldnot be required to find a rare binding site. Similarly, a cluster oftilted droplets in a sea of aligned droplets can be detected and, indoing so, would be able to detect a single analyte.

Detection of Agglutinated Janus Droplets

The solution of Janus droplets generally turns from transparent toopaque when the emulsions are agglutinated. FIG. 5A shows a solution ofJanus droplets before exposure to an analyte. FIG. 5B shows a solutionof Janus droplets after exposure to the analyte. Such large and easilyobservable differences may be incorporated into the use of imageprocessing algorithms to analyze the optical micrographs. These opticalmicrographs are readily taken from, for example, any common smartphoneequipped with magnifying lenses to enable low-magnification of 4× and10× (FIG. 6A).

For qualitative purposes, the detection may use the significant changesin the optical transparency between pristine and agglutinated Janusdroplets to generate a binary response. For example a transparentanalysis chamber containing the Janus droplets was placed on top of atwo-dimensional QR code, as shown in FIG. 6B. In the presence of ConA,the chamber became opaque and covered a portion of the QR code. Thistransformation inhibited a smartphone from reading the QR code.

To quantify the degree of agglutination, an image processing programthat calculates the percentage of area covered by agglutinated Janusdroplets by two distinct logics was implemented: 1) the amount ofoverlapping droplets and 2) the difference in optical intensity of theimages. FIGS. 7A-7C show the quantification of a plurality of Janusdroplets in the absence of a targeted analyte. FIGS. 7D-7F show thequantification of a plurality of Janus droplets exposed to a targetedanalyte.

Specifically, the image processing program analyzed the raw opticalmicrographs (FIG. 7A and FIG. 7D) by mapping out the locations of eachJanus droplet and measuring their radii (FIG. 7B and FIG. 7E). Usingthis information, the program then sought overlapping emulsions. Asdescribed above, during agglutination the Janus droplets joined togetherto form droplet complexes of agglutinated Janus droplets. The programdistinguished each droplet with more than two overlapping neighbors as apart of a droplet complex and rejected any droplet with zero, one, ortwo overlapping neighbors (FIG. 7C and FIG. 7F). The percentage of areacovered by agglutinated Janus droplets were then calculated for bothpristine sample (FIG. 7C) and agglutinated sample (FIG. 7F).

The area covered by these Janus droplet agglutinations were then furthercorrelated with the analysis of optical intensity within the images.Similar to the qualitative detection, the image analysis can distinguishregions of agglutinated Janus droplets due to the lower opticaltransparency. The program used an adaptive thresholding algorithm todistinguish areas with higher transparency (pristine Janus droplets)from the opaque regions (agglutinated Janus droplets), FIGS. 8A-8F. Thecombination of the two distinct logics—identifying the overlapping Janusdroplets and analyzing changes in optical intensity—can accuratelydetect the regions of agglutinated Janus droplets. Furthermore, thewhole process can be completed within seconds from capturing the imageto final calculation.

In some cases, the Janus droplets behave as individual lenses. Suchdroplets can be interrogated with a scanning light beam or a number ofbeams simultaneously. In this case (e.g., FIGS. 8A-8F), the light beamstransmit through the sample and impinge on an array of light detectors.Signals can be deduced by changes in the intensity that represents thestraight path of the light beam and the light that is refracted (e.g.,deviating from a straight path). Without wishing to be bound my theory,lower intensity at the point of the straight path and higher intensityof light that is refracted from that path, indicate an increase in thetilt of one or more droplets. Similarly, higher intensity of light inthe straight path and lower intensity that has been refracted mayindicate a decrease in the tilt of the droplet. Such lensing permitsdetection of changes in a single droplet. For example, the ability todetect single events that can lead to the detection of single pathogens,cells, catalysts, or molecules.

FIG. 9 details a strategy wherein breaking a single linkage (tether) canpotentially generate a sensor response that is visible to the naked eye.In this system, the red phase of the Janus droplet (CSC) had a higherdensity, and a gravitational force worked to orient the particles.Disrupting a chemical bond or complementary DNA interaction tehter,which has pinned the Janus droplet in a tilted scattering configuration,produced a relaxation to the transmissive equilibrium orientation. Anadvantage of this method is, for example, that only one droplet in amultitude of droplets need be rotated to be detected. Additionally bytethering to patterned surfaces, arrays of sensors can be produced thatcan detect multiple types of analytes in a single device.

Formation of Droplets

Materials. For the detection of ConA, hexane and perfluorohexane werechosen as the hydrocarbon and fluorocarbon phases respectively. In othercases, different pairs of hydrocarbon (ortho-dichlorobenzene, phthalate,etc.) and fluorocarbon (ethyl nonafluorobutyl ether, methoxyperfluorobutane, etc.) phases can be substituted to tune the uppercritical temperature (T_(a)) of the mixture and the differences indensity for suitable applications. For the continuous water phase,surfactants ManC14 and Zonyl® FS 300 were chosen to stabilize andgenerate the Janus droplets. The two surfactants were dissolved in HEPESbuffer solution (pH=7.5) separately with concentration of 0.0005% and0.01% by weight, respectively. In both bulk emulsification andmicrofluidics method, the final volume ratio between ManC14 solution andZonyl FS 300 solution was kept at 1.2:1 to generate two-hemisphere Janusdroplets. For surfactants that are soluble in water (such as ManC14 andZonyl® FS 300), a solution containing the functionalized surfactants wasused as the continuous phase.Bulk emulsification for polydispersed Janus droplets. To generate Janusdroplets via bulk emulsions, we began by preparing an equal-mixture ofhexane and perfluorohexane with a total volume of 1 mL in a 5 mL glassvial. The mixture initially formed an immiscible solution at roomtemperature. The vial containing the mixture was then heated to abovethe T_(c) using a standard heat gun until the mixture was miscible; forhexane-perfluorohexane mixture, the T_(c) is 20° C. For othercombinations of hydrocarbon and fluorocarbon, the T_(c) may varydepending on the two liquids. In another 5 mL glass vial, 1 mL of thecontinuous phase containing ManC14 and Zonyl FS 300 (concentrations ofboth reported in the previous section) was also heated to the sametemperature as the vial containing hexane-perfluorohexane mixture. Thisprecaution may mitigate the phase segregation of hexane andperfluorohexane upon addition before emulsification. 50 uL of heated andmiscible hexane-perfluorohexane mixture was then injected into theheated continuous phase via a pipette. The Janus droplets were thengenerated by shaking the vial using a vortex mixer at 3000 RPM for 5seconds. The solution of Janus droplets was then cooled down below T_(c)using an ice bath. This method of bulk emulsification generatedpolydispersed droplets with diameters ranging from 30 to 200 μm asobserved by an optical microscope.Generation of monodispersed Janus droplets via microfluidics. Bothcoaxial glass capillary microfluidics and commercial availablemicrofluidic chips were used to generate emulsions. For coaxial glasscapillary microfluidics, devices were made from an outer squarecapillary (OD=1.5 mm, ID=1.05 mm, AIT Glass) and inner cylindricalcapillary (OD=1 mm, World Precision Instruments) pulled to a 30 μm tipusing a P-1000 Micropipette Puller (Sutter Instrument Company). Forcommercial microfluidic device, Focused Flow Droplet Generator chip(channel width=100 μm, channel depth=20 μm, tip width=10 μm, glass) fromMicronit was used. In both microfluidics system, Harvard Apparatus PHDUltra syringe pumps were used to inject the outer phase (continuousphase) and inner phase (droplet phase). The flow rates were 50 μL min⁻¹for the continuous phase and 30 μL min⁻¹ for the droplet phase. Thesolution of monodispersed droplets was first collected via 20 mL glassvial and later diluted with both ManC14 solution and Zonyl® solution toachieve a final droplet phase concentration of 6% by volume whilemaintaining the 1.2:1 volume ratio of the two surfactants. Themicrofluidic setup was heated above the T_(c) of the inner phasesolution using a heat lamp. Janus droplets were then cooled below T_(c)to induce phase separation. For hexane-perfluorohexane emulsions, theemulsions were chilled on ice prior to imaging and often imaged whileimmersed in a cool water bath to maintain a temperature below 20° C. Theaverage diameter of the monodispersed droplets generated from this setupwere 60±10 μm. The composition of each droplet was nearly identicalbecause each droplet was generated from the same single droplet phase.Stability and sample storage. The Janus droplets generated from eithermethod described above were observed to be stable on the order of weeksunder room temperature. After emulsification, the Janus droplets werekept within the continuous phase at room temperature in a closed glassvial without mechanical perturbation. The diameter of the Janus dropletswas not observed to change significantly after weeks of storage.

Sensing

Sample preparation for sensing of ConA. Monodispersed or polydispersedJanus droplets used for sensing experiments were fabricated usingmethods described above. Janus droplets were loaded into a stainlesssteel sample holder with a 1 cm deep well and a 1.5 cm diameter viewingwindow. 0.5 mL of mixed surfactant solution containing 30 μL ofhexane-perfluorohexane droplet phase was loaded into sample holder tocreate a monolayer of Janus droplet that covered the whole viewingwindow. The sample holder and solution of the Janus droplets were keptbelow 20° C., the T_(c) of hexane-perfluorohexane mixture, during thesensing of ConA and image acquisition.Model system: Sensing of ConA. ConA was dissolved in HEPES buffersolution with final concentration of 0.5 mg mL⁻¹ and used as theanalyte. 10 μL, of ConA solution was added using a micropipette to thesample holder containing Janus droplets. Solution was then swirledgently and agglutination of Janus droplets were observed within seconds.Image were recorded before and after adding ConA solution. An increasingvolume (up to 40 μL) of ConA solution were added afterwards to get acorrelation between agglutination level and analyte concentration.Agglutination level were analyzed both qualitatively and quantitativelyas described below.

Surface Chemistry

Fabrication of DNA functionalized surface. Glass substrates were cleanedby sonication in acetone and isopropyl alcohol for 5 min each to removedust. After drying completely, the glass substrates were immersed inpiranha solution (H₂SO₄:H₂O₂, 1:1, v/v) for 1 h, rinsed thoroughly withdistilled water, and then dried under N₂. The glass substrates were thenimmersed and reacted with a toluene solution of trichlorosilane linkerterminated with an N-hydroxysuccinimide (NHS) for 1 h to form NHScovalently functionalized glass substrates. Afterwards, a solution of 10μM ssDNA dissolved in a sodium tetraborate buffer at pH 9 was reacted toform an amide bond, which attach the ssDNA onto surface of the glassslides. ssDNA was functionalized with alkyl chain to form a surfactantmolecule. Janus droplets residing on the surface of ssDNA functionalizedglass substrate were tilted against gravity. A solution of thecomplementary strand dissolved in 0.25 M NaCl solution was added toJanus droplets to hybridize the DNA strands. Janus droplets werereleased from the glass substrate to be aligned with gravity at areaswhere DNA strands were hybridized. X-ray photoelectron spectroscopy wasused to analyze the elements on glass substrates to ensure successfulfunctionalization of ssDNA.

Detection

Sample preparation for detection. For both qualitative and quantitativemethods of detection, Janus droplets were imaged in a stainless steelsample holder. For qualitative detection, a two-dimensional QR code (1cm×1 cm) was placed 1 cm below the viewing window of the analysischamber. For quantitative detection, a white background was used insteadof the QR code to provide contrast. The analysis chamber and thesolution of the Janus droplets were kept in an ice bath, well below theT_(c) of the hexane-perfluorohexane mixture to maintain the morphologyof the Janus droplets.Qualitative analysis using QR code. Qualitative analysis was performedusing the QR code from unmagnified images taken from the smartphone. Thedistance from the phone to the analysis chamber containing the Janusdroplets was approximately 10 cm. The exact distance was calibrated bythe image processing software by using the known dimension of the QRcode (1 cm×1 cm). The binary response measured was whether the QR codecould be read via the software. If the QR code was readable, the Janusdroplets were considered not agglutinated, and vice versa.Image acquisition for quantitative analysis. To acquire thelow-magnification images for quantitative analysis, an adaptor withmagnifying lenses was adapted onto the smartphone. With thismodification, optical micrographs with 4× and 10× magnification wereobtained. The working distance from the smartphone to the analysischamber was 1 cm. The working distance and the dimension of the imageswere calibrated by the calibrated marking underneath the analysischamber with 10 μm tick marks. The image processing software thenpre-processed the captured images by transforming them into greyscaleimages and adjusting the brightness and contrast to the reference imageof blank analysis chamber. For each sample, 100 pictures were taken,forming a 10×10 array of images to span the majority of the area of theanalysis chamber.Identification of overlapping Janus droplets. From the pre-processedimages with 10× magnification (greyscale images with adjusted brightnessand contrast), the image processing program first estimated the range ofdiameters of the Janus droplets by using the calibrated markingunderneath the analysis chamber. The program then sought out and mappedthe centers and calculated the diameters of every Janus droplet. Thisprocess was done by a modified method based on the Circle HoughTransform. With the coordinates of the centers and the diameters of theJanus droplets, the program then evaluated overlapping droplets.Specifically, if the distance between two centers of two droplets wassmaller than the sum of the two radii, the droplets were consideredoverlapping. Using this logic, the program could effectively map out thenumber of overlapping neighbors for every identified droplet.Identification of droplet complexes. A Janus droplet was considered tobe a part of a droplet complex if the number of its overlapping neighborexceeded three. This threshold was set in some cases to preventover-counting of the droplets at the edges of the droplet complexes andaccidental overlapping of droplets. This measurement was furthercollaborated by the analysis based on the optical intensity. The areaoccupied by the agglutinated droplet complexes was then calculated.Analysis of changes in optical intensity. Using the pre-processed imagesof 4× magnification (greyscale images with adjusted brightness andcontrast), the program first applied the adaptive thresholding algorithmto distinguish the darker edges of the Janus droplets from the dropletcomplexes with tilted particles. More specifically, the program ignoredthe edges of the droplets that have inherent low-light intensity andonly sought the area of droplet complexes. A threshold was set usingareas with light intensity of less than 45% of the brightest regions tobe considered part of the droplets complex. From this information, thearea occupied by the droplet complexes was then calculated.

Detection of Zika Virus

Zika is a vector-borne flavivirus which has emerged as a global healthpriority in recent years. Although Zika virus infections typically onlycause mild febrile symptoms in adults, the virus can be passed frominfected pregnant women to their fetuses and has been linked to severebirth defects such as microcephaly. Additionally, Zika virus has beenconnected to neurological disorders in adults, includingGuillain-Barr-Syndrome. No approved vaccines or treatments currentlyexist for Zika virus; as a consequence rapid accurate detection of Zikavirus is essential to control epidemics and reduce the risk of theseneurological complications. In recent years, many researchers havefocused on developing assays for the detection of Zika virus, includingpolymerase chain reaction (PCR) and antibody based assays. Additionally,Zika virus detection using RNA amplification and CRISPR/Cas9 in rapidand low-cost sensors has been reported to be used in pandemic regions.However, there is still a need for a sensing assay with high stability,lower cost, and less reliance on specialized instrumentation, whichcould become essential in areas with endemic transmission of Zika.

This example is generally related to the use of Janus for the detectionof the Zika virus. In this example, detection is conducted viarecognition of protein NS1, a non-structural hexameric biomarker whichplays a role in pathogenesis and immune evasion, although other proteinsare also possible. This agglutination assay for the detection of thisanalyte that is generally robust, low cost, and readily multiplexed.

Selectivity and sensitivity in this emulsion agglutination assay isgenerally determined by the specific binding activity to the targetanalyte and the translation of analyte binding to an agglutinatedcomplex, which in turn changes optical transmission through a layer ofemulsion droplets. To conjugate thiol containing receptor biomolecules,a maleimide-functionalized polystyrene-b-polyacrylic acid (P1-MA) wasemployed as the surfactant. Using this construction variants of thehyperthermophilic binding protein were conjugated, reduced-charged Sso7d(rcSso7d). This selection is attractive as the rcSso7d protein is aviable replacement for antibodies in immunoassays, as a result of itsintrinsic thermal and chemical stability, ease of large-scalebiomanufacturing, and a versatile binding face. This protein scaffoldcan further be engineered to have high affinity for specific targetproteins. To optimize an agglutination assay, the streptavidin-bindingrcSso7d variant (rcSso7d-SA) was used for the protein-proteinrecognition. In addition, two novel optical transduction methods, whichcan be readily instrumented, were prepared for the quantification of theanalyte. This optimization allowed for the efficient integration of thercSso7d Zika NS1 binding variant (rcSso7d-ZNS1) into the assay format.The optimized system demonstrates a detection limit of 100 nM for theZika NS1 protein. Emulsion droplet disposable assays based on theconstructions presented herein have the advantage of thermally stablerecognition elements, simple detection, and the avoidance of nucleicacid extractions that often require a trained technician.

Gallic acid derived surfactants may be used for droplet bioconjugation.These surfactants exhibited good stability and displayed sensor behaviorby triggering droplet morphology changes. However, the gallic acidderived surfactants may not, in some cases, have the anchoring strengthnecessary to hold multiple droplets together in an agglutination assay.The block co-polymer anchor, polystyrene-b-polyacrylic acid (P1)displayed a good connection strength between the protein and droplets.The acrylic acid block in P1 was functionalized with maleimide-NH2 toform P1-MA (FIG. 10A). P1-MA was dissolved in the hydrocarbon phase andbehaves as a hydrocarbon/water (H/W) interfacial active agent. Withoutwishing to be bound by theory, this generally positions the maleimidegroup at the H/W interphase, enables the maleimide-thiol bioconjugationof cysteine-bearing proteins, and the production ofprotein-functionalized emulsion droplets.

To produce agglutination with the emulsion droplets, protein-proteininteractions are used, wherein the recognition protein rcSso7d isimmobilized on the droplets. For this construction, the previouslyengineered rcSso7d-SA was further genetically modified with a cysteineresidue on the N-terminus for bioconjugation to the droplet. Thecysteine-modified rcSso7d-SA was then covalently linked to thehydrocarbon-water (H/W) interface via a maleimide-thiol conjugateaddition reaction. The addition of the tetravalent streptavidin to thercSso7d-SA functionalized droplets triggers agglutination by linkingrcSso7d from different droplets together (FIG. 10B). The agglutinationwas observed within 30 min of the addition of the streptavidin.

The amount of rcSso7d-SA conjugated to the droplet H/W interface isgenerally related to droplet morphology during the reaction. To optimizethe level of functionalization of rcSso7d-SA for agglutination, threeinitial morphologies (1) hydrocarbon-in-fluorocarbon-in-water (H/F/W),(2) Janus, and (3) fluorocarbon-in-hydrocarbon-in-water (F/H/W) werecreated by tuning the proportions of the continuous phase surfactants.As shown in FIG. 11, each droplet initially has a different morphologyand consequently a different surface area at the H/W interface forbioconjugation. After rcSso7d-SA functionalization, each droplet typewas switched to the same morphology (Janus state) by exchanging thecontinuous phase surfactant solution in order to compare the relativeagglutination responses. Streptavidin (0.36 μM) was added to agglutinatethe Janus droplets. As shown in the micrographs in FIG. 11, the dropletsthat were functionalized in a F/H/W morphology displayed higheragglutination levels. This result indicates that the increased surfacearea at the hydrocarbon/water interface facilitates thefunctionalization with rcSso7d and increases the agglutinationsensitivity. Although a higher concentration of Tween 20 is used for theF/H/W morphology, the P1-MA molecule exhibits stronger surfactantbehavior and effectively competes to partition at the H/W interface foran optimal maleimide-thiol conjugation reaction. Given the greaterdegree of agglutination achieved using droplets conjugated in the F/H/Wmorphology, this configuration was used for rcSso7d functionalization inall of the following studies.

Having established this emulsion assay for the detection of smallquantities of streptavidin, a quantitative optical read-out of dropletagglutination was created. Without wishing to be bound by theory,Multi-compartment colloids have intrinsic optical properties as a resultof the differing refractive indices of the constituent phases. Doubleemulsion droplets are tuneable lenses with optical properties that varywith the droplet morphology. The refractive index contrast and curvatureof each interface contribute to the lenses' ability to focus or scatter.The strong variations in the light transmission properties of Janusdroplets before and after agglutination are displayed in the opticalmicrographs of FIG. 11. Increases in the fraction of agglutinated(tilted) droplets causes more scattering of the incoming light, whichcan be measured in transmission or backscattering modes normal to thedroplet layer. To produce a system displaying large optical effects, wechose a solvent combination with a relatively large refractive index(RI) contrast, diethylbenzene (RI: 1.49) for the hydrocarbon phase andHFE 7500 (RI:

1.29) for the fluorocarbon phase. Depending on the angle of the internalH/F interface, the incoming light rays can intersect the internal H/Finterface at angles below or above the critical angle: θ_(c)=60°. As aresult, depending on the droplet's morphology and the refractive indexcontrast, the incoming light could undergo total internal reflection(TIR). In this agglutination sensing scheme, the selectors wereselectively immobilized at the hydrocarbon/water interphase, which leadsto the aggregation of the high RI hydrocarbon phases. As a result of thesurfaces and RI contrast, in the agglutinated state light can bebackscattered in the upward direction, similar to a corner cubereflector (FIG. 12B and FIG. 15). In order to translate this lightscattering effect into an applicable optical detection scheme, anoptical fiber was positioned on top a droplet monolayer and recorded thelight intensity (FIG. 12A). For reproducibility and for creating aratiometric read-out of the degree of agglutination, a fluorescent dye(perylene; 1.5 mM) was added to the hydrocarbon phase and the intensityratio of the excitation light (I_(exc)) to the reference peryleneemission (I_(H)) was recorded. Increased tilting of the droplets leadsto an increase of the backscattered light intensity. Depending on theconcentration of streptavidin and therefore the degree of agglutination,this optical sensor scheme provides a ratiometric signal forquantitative measurements with maximum intensity increase of up to 50%(FIG. 12C).

In addition, an additional purely fluorescent based sensor read-out ofdroplet agglutination was created. This method provides a robust scheme,with the advantage of using multilayers of polydisperse droplets, moreaccurate ratiometric signals, and the possibility of multiplexing. Inthis scheme a second emissive dye (F-PDI) was dissolved in thefluorocarbon phase. Changes in droplet alignment in response tostreptavidin were quantified by recording the ratio of the emission fromtwo dyes in the hydrocarbon and the fluorocarbon phase (FIG. 13A). Bytargeted selection of the dyes, the emitted light of one of the dyes canbe selectively attenuated via the inner filter effect depending on theorientation of the droplets. F-PDI exhibits absorption with a spectraloverlap with the emission of a high band gap (blue light emitting)perylene dye in the hydrocarbon phase. The overall emission of theemulsion may be dominated, in some cases, by the red fluorophore if thedroplets are arranged in their gravity aligned fashion because theemitted light from the perylene dye collected by the optical fiber hasto pass through the phase with the F-PDI dye before exiting the droplet.The degree of agglutination is accompanied by a continuous increase ofthe emission of the perylene dye, as a result of the decreased pathlength though the absorbing fluorocarbon phase (FIG. 13B). As displayedin FIG. 13C, the detection of the ratio of perylene (I_(H)) and F-PDI(I_(F)) emission provide for precise correlation with the level ofagglutination, which can be used to quantify analyte concentration.Similar to the backscattering scheme described above, higherconcentrations of streptavidin produce saturation of the light intensityratio. Both methods show similar analyte sensitivity, which is indicatedby the slope of the curve at low concentration.

The agglutination assay was then adapted for the detection of thepolyvalent Zika NS1 protein using droplets conjugated to rcSso7d-ZNS1via a cysteine at the N-terminus. The intensity curves reveal thesensitivity of the agglutination assay for Zika NS1 and the results areshown in FIG. 14. As a result of the lower binding strength of the ZikaNS1 to the rcSso7d-ZNS1 as compared to the streptavidin to rcSso7d-SA,the reaction was equilibrated overnight, which allows for higheragglutination yields and the lowest limit of detection. The two opticalschemes using the backscattering and inner filter effect are inagreement after normalization to the maximum (saturated) emissionintensity, with a limit of detection of 100 nM.

In summary, an agglutination assay for the sensing of Zika NS1 proteinat a limit of detection of 100 nM using two optical schemes that can beeasily prototyped was described. A maleimide functionalized P1-MApolymer was used as the surface active agent to covalently link thehyperthermophilic rcSso7d protein to the surface of the droplets. Themultivalent protein analyte Zika NS1 binds to surface bound rcSso7dgroups to cause droplet agglutination. Robust ratiometric signals todetect agglutination were developed by incorporating dyes in thedroplets and detecting backscattered/emitted light, or multipleemissions modulated by an inner filter effect. This emulsionagglutination assay offers low power requirements without complicatedlabelling and nucleic acid handling. The assay is potentially suitablefor use in remote locations without access to expensive equipment andtrained personnel to identify Zika virus infections as well as otherpathogenic species.

While several embodiments of the present invention have been describedand illustrated herein, those of ordinary skill in the art will readilyenvision a variety of other means and/or structures for performing thefunctions and/or obtaining the results and/or one or more of theadvantages described herein, and each of such variations and/ormodifications is deemed to be within the scope of the present invention.More generally, those skilled in the art will readily appreciate thatall parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the teachings of thepresent invention is/are used. Those skilled in the art will recognize,or be able to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. It is, therefore, to be understood that the foregoingembodiments are presented by way of example only and that, within thescope of the appended claims and equivalents thereto, the invention maybe practiced otherwise than as specifically described and claimed. Thepresent invention is directed to each individual feature, system,article, material, kit, and/or method described herein. In addition, anycombination of two or more such features, systems, articles, materials,kits, and/or methods, if such features, systems, articles, materials,kits, and/or methods are not mutually inconsistent, is included withinthe scope of the present invention.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Other elements may optionallybe present other than the elements specifically identified by the“and/or” clause, whether related or unrelated to those elementsspecifically identified unless clearly indicated to the contrary. Thus,as a non-limiting example, a reference to “A and/or B,” when used inconjunction with open-ended language such as “comprising” can refer, inone embodiment, to A without B (optionally including elements other thanB); in another embodiment, to B without A (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” and the like are to be understoodto be open-ended, i.e., to mean including but not limited to. Only thetransitional phrases “consisting of” and “consisting essentially of”shall be closed or semi-closed transitional phrases, respectively, asset forth in the United States Patent Office Manual of Patent ExaminingProcedures, Section 2111.03.

Any terms as used herein related to shape, orientation, alignment,and/or geometric relationship of or between, for example, one or morearticles, structures, forces, fields, flows, directions/trajectories,and/or subcomponents thereof and/or combinations thereof and/or anyother tangible or intangible elements not listed above amenable tocharacterization by such terms, unless otherwise defined or indicated,shall be understood to not require absolute conformance to amathematical definition of such term, but, rather, shall be understoodto indicate conformance to the mathematical definition of such term tothe extent possible for the subject matter so characterized as would beunderstood by one skilled in the art most closely related to suchsubject matter. Examples of such terms related to shape, orientation,and/or geometric relationship include, but are not limited to termsdescriptive of: shape—such as, round, square, circular/circle,rectangular/rectangle, triangular/triangle, cylindrical/cylinder,elliptical/ellipse, (n)polygonal/(n)polygon, etc.; angular orientation -such as perpendicular, orthogonal, parallel, vertical, horizontal,collinear, etc.; contour and/or trajectory—such as, plane/planar,coplanar, hemispherical, semi-hemispherical, line/linear, hyperbolic,parabolic, flat, curved, straight, arcuate, sinusoidal,tangent/tangential, etc.; direction—such as, north, south, east, west,etc.; surface and/or bulk material properties and/or spatial/temporalresolution and/or distribution—such as, smooth, reflective, transparent,clear, opaque, rigid, impermeable, uniform(ly), inert, non-wettable,insoluble, steady, invariant, constant, homogeneous, etc.; as well asmany others that would be apparent to those skilled in the relevantarts. As one example, a fabricated article that would described hereinas being “square” would not require such article to have faces or sidesthat are perfectly planar or linear and that intersect at angles ofexactly 90 degrees (indeed, such an article can only exist as amathematical abstraction), but rather, the shape of such article shouldbe interpreted as approximating a “ square,” as defined mathematically,to an extent typically achievable and achieved for the recitedfabrication technique as would be understood by those skilled in the artor as specifically described. As another example, two or more fabricatedarticles that would described herein as being “ aligned” would notrequire such articles to have faces or sides that are perfectly aligned(indeed, such an article can only exist as a mathematical abstraction),but rather, the arrangement of such articles should be interpreted asapproximating “aligned,” as defined mathematically, to an extenttypically achievable and achieved for the recited fabrication techniqueas would be understood by those skilled in the art or as specificallydescribed.

1-40. (canceled)
 41. A system comprising: a plurality of Janus dropletsassociated with a plurality of binding moieties to a virus; and adetector positioned relative to the plurality of Janus droplets suchthat when sufficient numbers of the binding moieties bind to the virusat least a portion of the plurality of Janus droplets are changed inorientation sufficient to change electromagnetic radiation interactingwith the Janus droplets in a manner determinable by the detector.
 42. Amethod for determining the presence of a virus, comprising: exposing, toan article comprising an outer phase and a plurality of Janus dropletsdispersed within the outer phase, a sample suspected of containing thevirus, wherein the virus, if present, interacts with at least a portionof the article such that at least a portion of the plurality of Janusdroplets change orientation thereby producing a detectable change in anoptical property of the article; and determining the detectable change.43. A system as in claim 40, wherein each Janus droplet comprises afirst phase and a second phase, immiscible with the first phase.
 44. Asystem as in claim 40, wherein the outer phase is an aqueous phase. 45.A system as in claim 40, wherein the first phase comprises ahydrocarbon, a fluorocarbon, a silicone, a liquid crystal, an ionicliquid, a polymer, combinations thereof, or derivatives thereof.
 46. Asystem as in claim 40, wherein the second phase comprises a hydrocarbon,a fluorocarbon, a silicone, a liquid crystal, an ionic liquid, apolymer, combinations thereof, or derivatives thereof, immiscible withthe first phase.
 47. A system as in claim 40, wherein the amphiphiliccompound is selected from the group consisting of: ionic surfactants,non-ionic surfactants, zwitterionic surfactants, polymers, blockcopolymers, proteins, DNA, RNA, acids, carbohydrates, saccharides,enzymes, chromophores, lipids, graphene oxide, combinations thereof,nanoparticles, and derivatives thereof.
 48. A system as in claim 40,wherein an interface between the outer phase and the plurality of Janusdroplets comprises the amphiphilic compound.
 49. A system as in claim40, wherein upon binding to the binding moieties, at least a portion ofthe plurality of Janus droplets agglutinate.
 50. A system as in claim40, wherein, prior to binding to the binding moieties, the plurality ofJanus droplets are oriented such that at least a portion of interfacesbetween a first phase and a second phase within each Janus droplet arealigned parallel with respect to one another.